This study is engaged in the detection and characterization of transient oxygenated intermediates which occur in enzyme-catalyzed reactions. The reactions of molecular oxygen and partially reduced oxygen (H2O2 and superoxide) with several redox-active enzymes will be investigated by rapid reaction techniques including stopped-flow optical spectrophotometry and rapid freeze-quenching combined with Mossbauer and EPR spectrometry. The enzymes to be studied are: liver microsomal P-450LM monooxygenases and erythrocyte formyl-containing heme protein as examples of hemoproteins; bacterial parahydroxybenzoate hydroxylase and a mammalian microsomal drug oxidase as example of flavoproteins; and bacterial protocatechuate dioxygenase as an example of a nonheme iron-containing protein. It is hoped that some general requirements and properties associated with the mechanisms for the biological activation of oxygen will be learned during the course of these studies. Work utilizing many of the same techniques will be done on the flavin disulfide enzyme, lipoyl dehydrogenase, to try and further understand the mechanism of the enzymatic interchange of electrons from reduced pyridine nucleotides to disulfides. Work is also planned on some of the kinetic properties of prostaglandin synthetase. Many of the above studies will be performed in collaboration with the following investigators and their colleagues: Drs. Vincent Massey, M.J. Coon, D.E. Hultquist, R.G. Matthews, and C.H. Williams (all Department of Biological Chemistry) and Dr. D.M. Ziegler, Clayton Foundation Biochemical Institute, University of Texas at Austin. BIBLIOGRAPHIC REFERENCES: Matthews, R.G., Ballou, D.P., Thorpe, C., Williams, C.H. (1977) Ion Pair Formation in Pig Heart Lipoamide Dehydrogenase, J. Biol. Chem. 252, 3199-3207. Coon, M.J., White, R.E., Nordbloom, G.D., Ballou, D.P., Guengerich, F.P. (1977) Highly Purified Liver Microsomal Cytochrome P-450; Properties and Catalytic Mechanism, Croatia Chemica Acta 49, 163-177.